200 nm polystyrene beads

For the Flow Nano Analyzer (NanoFCM Co. Ltd., Nottingham, UK) analysis, the system was calibrated using 200 nm polystyrene beads (NanoFCM Co. Ltd., Nottingham, UK) with a defined concentration of 5.7 × 10⁸ particles/mL, which were also used as a reference for the particle concentration. In addition, monodisperse silica beads (NanoFCM Co. Ltd., Nottingham, UK) of four different sizes were used as reference standards to calibrate the size of the EVs. Freshly filtered (0.22 µm) PBS was analyzed as a background signal, which was subtracted from the other measurements. EV samples were diluted with filtered PBS resulting in a particle count in an optimum range of 2500–12,000 events, and sample data were collected for 1 min with a sample pressure of 0.4 kPa. Particle concentration and size distribution were calculated using the NanoFCM software NF Profession v1.08) (NanoFCM Co. Ltd., Nottingham, UK). Median and interquartile range (IQR) were calculated using R software v4.1.0 (R Foundation, Vienna, Austria).

For nanoFCM, a Nano Analyzer (NanoFCM Co. Ltd., Nottingham, United Kingdom) equipped with a 488 nm laser, was calibrated using 200 nm polystyrene beads (NanoFCM Co.) with a defined concentration of 2.08 × 108 particles/ml, which were also used as a reference for particle concentration. In addition, monodisperse silica beads (NanoFCM Co. Ltd.) of four different sizes served as size reference standards to calibrate the size of EVs. Freshly filtered (0.1 µm) 1 × PBS was analyzed as background signal and subtracted from the other measurements. Each dot plot was derived from data collected approximately 4,000 events with a sample pressure of 1.0 kPa. For immunofluorescence staining, the following antibodies were used (BioLegend): FITC-conjugated mouse anti-human/canine CD9 antibody (clone HI9a), and anti-human Ecad, with secondary PE-conjugated donkey anti-rabbit IgG antibody (clone Poly4064); as isotype controls, FITC-conjugated mouse IgG1, κ (clone MOCP-21), and PE-conjugated donkey anti-rabbit IgG antibody (clone Poly4064); 1 ng/µl of each antibody in 50 µL 1 × PBS. After removing antibody aggregates by centrifugation at 12,000× g for 10 min, the supernatant was added to 5 × 108 purified EVs, followed by incubation for 90 min at 25°C under constant shaking. Stained EV were diluted 1:100 in 1xPBS for NanoFCM analysis.

For nFCM a Nano Analyzer (NanoFCM Co., Ltd, Nottingham, UK) equipped with a 488 nm laser was calibrated with 200 nm polystyrene beads (NanoFCM Co.) with a defined concentration of 2.08 × 10^8 particles/ml and also used as a reference for particle concentration. In addition, monodisperse silica beads (NanoFCM Co.) of four different sizes were used as a size reference standard to calibrate the size of the vesicles. Freshly filtered (0.1 µm) 1 × PBS was analyzed as background signal and subtracted from the other measurements. Each distribution histogram or dot plot was derived from data collected for 1 min with a sample pressure of 1 kPa. OMV samples were diluted with filtered (0.1 µm) 1 × PBS, resulting in particle counts in the optimal range of 2,500–12,000 events. Particle concentration and size distribution were calculated using nFCM software (NF Profession V1.08).